Ti-4 protein derived from Triatoma infestans exhibiting activity to inhibit platelet aggregation

ABSTRACT

The present invention provides Ti-4 protein, which is a protein obtained from salivary gland of  Triatoma infestans , and the present invention provides Ti-4 gene encoding the protein. As the Ti-4 protein exhibits inhibitory activity on platelet aggregation, a medicine comprising the Ti-4 protein as an active ingredient will serve as a platelet aggregation inhibitor possibly effective for prevention and treatment of myocardial infarction, pulmonary infarction and cerebral infarction. Moreover, the Ti-4 protein will also serve as a highly prospective lead compound in the development of novel platelet aggregation inhibitors.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to Ti-4 protein, which is a proteinderived from salivary gland of Triatoma infestans (an assassin bug)exhibiting activity to inhibit platelet aggregation, and to a geneencoding the Ti-4 protein.

2. Description of the Related Art

With progression of aging of society, treatment of adult diseases as asocial problem is becoming more important. What is the most importantfor the welfare of such aging society is treatment and prevention ofsymptoms related to adult diseases, particularly cardiovasculardisorders resulting from vascular sclerosis such as hypertension,pulmonary hypertension, myocardial infarction, cerebral infarction,pulmonary infarction and vascular spasms followed by subarachnoidhemorrhage. Such vascular disorders can be prevented and treated byadministration of vasodepressors, blood coagulation inhibitors andplatelet aggregation inhibitors to the patients. The known peptidehaving an anticoagulant activity which may be used as a medicine fortreating such vascular disorders includes hirudine, a peptide isolatedfrom salivary gland of leech. Hirudine is an anticoagulant peptideisolated from salivary gland of assassin bugs and it exhibitsanti-thrombic activity. Ti-4 protein of this invention and a geneencoding the protein are novel. However, general knowledge onphysiologically active substances isolated from salivary glands ofassassin bugs are described as reviews in the following scientificjournals:

-   (1) Ribeiro, J. M., “Role of saliva in blood-feeding by arthropods”,    Annu. Rev. Entomol., 1987, vol.32, p463-478-   (2) Basanova, A. V., Baskova, I. P., and Zavalova, L. L.,    “Vascular-platelet and plasma hemostatis regulators from    bloodsucking animals” Biochemistry, 2002, vol67, p143-150

However, hirudine has some problems when applied as a medicine, thoseare, synthesis of hirudine is difficult, and it has some adverseside-effects. To achieve large amount of production of the compound tobe used as a medicine in safely, it is necessary to solve the aboveproblems. Thus, isolation of a protein whose synthesis is easy andcapable of inhibiting the aggregation of platelets without causing anynotable side-effects has been needed. If such a protein were produced,it would be a useful lead compound in the production of ananticoagulant, and be highly promising in the development of newmedicines.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a protein exhibitingactivity to inhibit platelet aggregation isolated from salivary gland ofTriatoma infestans, a blood-sucking insect. Another object of thepresent invention is to provide a method to achieve production of theprotein in large amount, utilizing the system of baculovirus.

To achieve the above objects, the present application provides followinginventions. This invention provides Ti-4 protein derived from Triatomainfestans, consisting of an amino acid sequence represented by aminoacids Nos. (−22)-156 shown in SEQ ID No. 1 of the sequence listing.Other proteins consisting of an amino acid sequence in which a part ofabove-mentioned amino acid sequence is deleted, substituted, or anotheramino acid sequence is added to the above-mentioned amino acid sequenceare also included in this invention, so far as exhibiting the activityto inhibit platelet aggregation.

This invention further provides Ti-4 protein derived from Triatomainfestans, consisting of an amino acid sequence represented by aminoacids Nos. 1-156 shown in the SEQ ID No. 1 of the sequence listing.Other proteins consisting of an amino acid sequence in which a part ofabove-mentioned amino acid sequence is deleted, substituted, or anotheramino acid sequence is added to the above-mentioned amino acid sequenceare also included in this invention, so far as the exhibiting theactivity to inhibit platelet aggregation.

This invention further provides Ti-4 gene derived from Triatomainfestans, consisting of a base sequence represented by bases Nos. 1-644shown in the SEQ ID No. 2 of the sequence listing. Other genesconsisting of a base sequence in which a part of above-mentioned basesequence is deleted, substituted, or another base sequence is added tothe above-mentioned base sequence are also included in this invention,so far as encoding proteins exhibiting the activity to inhibit plateletaggregation.

Furthermore. this invention provides a platelet aggregation inhibitorcontaining the above-mentioned protein as an effective ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a figure showing the amino acid sequence of the Ti-4 protein,and the base sequence of the gene encoding the protein.

FIG. 2 is a graph illustrating the effect of Ti-4 protein oncollagen-induced platelet aggregation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The salivary glands derived from blood-sucking insects and mites containsubstances having specific activities toward blood or blood vessels.Therefore, the present inventors identified an active substance havinginhibitory effect to platelet aggregation, which is derived fromsalivary gland of such an insect. Then the inventors achieved isolationand purification of the substance, and further analyzed on theproperties of the active substance. In a subsequent study, the inventorsachieved cDNA cloning of the gene. Moreover, the inventors developed amethod for production of the protein having the activity to inhibitplatelet coagulation in a large scale, utilizing the system ofbaculovirus.

In concrete, the inventors paid attention to Triatoma infestans (Ti),which is a blood-sucking insect, and tried to isolate a proteinexhibiting inhibitory activity on platelet aggregation derived from theinsect. Salivary glands were removed from approximately 40 individualsof Triatoma infestans, total mRNA was extracted from the salivaryglands, dsDNA was synthesized using poly(A)⁺mRNA as a template byreverse transcriptase, then cDNA library of the salivary gland wasconstructed by inserting the dsDNA into a transfer vector. From cDNAlibrary of the Ti salivary gland, colonies were picked up at random andtheir base sequences were determined. The inventors sequenced on 550colonies, they picked up cDNAs containing secretion signal and excludedoverlapping cDNAs. As a consequence 44 cDNAs were obtained and totalbase sequences of them were determined. FIG. 1 shows base sequence ofthe Ti-4 gene thus obtained and amino acid sequence of the Ti-4 proteinencoded by the gene.

Out of them, transfer vector constructs were prepared on 16 cDNAs toachieve protein expression in an expression system using baculovirus(AcNPV). The viruses were transfected with the construct, and viralclones failing to form apocytes, namely clones expressing proteinsencoded by the inserts, were isolated. Expression of the protein wasconfirmed by SDS-PAGE, and the protein was isolated and purified by HPLCbased on gel filtration chromatography and ion exchange chromatography,then Ti-4 protein according to this invention was obtained. Then,investigation of the protein according to this invention thus obtainedwas performed on its effect toward platelet aggregation.

The substance of the target was added to washed human platelets, andcollagen was added to induce platelet aggregation. Ten minutes later,the light transmission of the sample was measured, and then inhibitoryeffect of the substance on the platelet aggregation was investigated. Asa consequence, the Ti-4 protein inhibited collagen-induced plateletaggregation in dose-dependent manner. From this result, it was concludedthat the Ti-4 protein according to this invention exhibited the effectto inhibit platelets aggregation, that is, the protein would beeffective as a platelet aggregation inhibitor. Therefore, the Ti-4protein will be effective as an active ingredient in medicines fortreatment or prevention of myocardial infarction, pulmonary infarctionand cerebral infarction.

The Ti-4 protein is defined by the amino acid sequence consisting ofamino acids Nos. (−22)-156 shown in the SEQ ID No. 1 of the sequencelisting. In this specification, the protein consisting of an amino acidsequence in which a part of said amino acid sequence represented SEQ IDNO: 1 in the sequence listing is deleted, substituted or added withanother amino acid sequence means a protein consisting of an amino acidsequence in which 20 or less, preferably 10 or less, and more preferably5 or less amino acids of the sequence is deleted, substituted or addedto the amino acid sequence represented by SEQ ID NO: 1 in the sequencelisting. Moreover, such protein exhibits homology 95% or more,preferably 97% or more and still preferably 99% or more with the aminoacid sequence represented by SEQ ID NO: 1 in the sequence listing. Suchpolypeptide is also within the range of this invention so far as itexhibits function as Ti-4 protein of inhibiting platelet aggregation.Meanwhile, in the SEQ ID NO: 1 in the sequence listing, the regioncorresponding to amino acids Nos. −22 to −1 represent a signal peptide.The peptide received processing to yield a mature protein consisting of156 represented by amino acids Nos. 1-156.

Moreover, the Ti-4 gene codes for the Ti-4 protein described above, andit consists of the base sequence represented by bases Nos. 1-644 shownin the SEQ ID No. 2 of the sequence listing. The region corresponding tobases Nos. 21-554 represents an open reading frame, and encodes theabove protein. According to technique of gene recombination, artificialmodification can be achieved at a specific site of basic DNA, withoutalteration or with improvement of basic characteristic of said DNA.Concerning a gene having native sequence provided according to thisinvention or modified sequence different from said native sequence, itis also possible to perform artificial modification such as insertion,deletion or substitution to obtain gene of equivalent or improvedcharacteristic compared with said native gene. Moreover, a gene withsuch mutation is also included in the range of this invention.

That is, the gene consisting of a base sequence in which a part of saidgene represented by base sequence represented by SEQ ID NO: 2 in thesequence listing is deleted, substituted or added with another basesequence means a gene consisting of a base sequence in which 20 or less,preferably 10 or less, and more preferably 5 or less bases of thesequence is deleted, substituted or added to the base sequencerepresented by SEQ ID NO: 2 in the sequence listing. Moreover, such geneexhibits homology 95% or more, preferably 97% or more and stillpreferably 99% or more with the base sequence represented by SEQ ID NO:2 in the sequence listing. Such gene is also within the range of thisinvention so far as it encodes a protein exhibiting function as Ti-4protein of inhibiting platelets aggregation. In addition, such genehybridizes with the base sequence represented by SEQ ID No.2 in thesequence listing under a stringent condition.

The condition for hybridization can be selected by a skilled artisan adlibitum. For example, hybridization can be performed by the followingprocedure. DNA molecules or RNA molecules to be tested are transferredonto a membrane, then the membrane is hybridized with a labeled probe ina proper hybridization buffer. The hybridization buffer may comprise,for example, 5×SSC, 0.1 (weight)% N-lauroylsarcosine, 0.02 (weight)%SDS, 2 (weight)% of blocking reagent for nucleic acid hybridization, and50% formamide. The blocking reagent for nucleic acid hybridization maycomprise, for example, a buffer (pH 7.5) containing 0.1M maleic acid and0.15M sodium chloride and commercially available blocking reagent forhybridization dissolved into the buffer at the concentration of 10%. The20×SSC solution may comprise 3M sodium chrolide and 0.3M citrate, andthe SSC solution may be preferably utilized at the concentration of 3 to6×SSC, more preferably at the concentration of 4 to 5×SSC.

The temperature for hybridization may preferably be 40 to 80° C., morepreferably be 50 to 70° C., further more preferably be 55 to 65° C.Incubation may be performed from several hours to overnight, then washedby a washing buffer. The temperature for washing may preferably be roomtemperature, more preferably it may be the temperature used forhybridization. The formulation for the washing buffer may preferablycomprise 6×SSC and 0.1% (weight %) SDS, more preferably may comprise4×SSC and 0.1% (weight %) SDS, further preferably may comprise 2×SSC and0.1% (weight %) SDS, more further preferably may comprise 1×SSC and 0.1%(weight %) SDS, most preferably may comprise 0.1×SSC and 0.1% (weight %)SDS. The membrane may be washed by such washing buffer, then DNAmolecule or RNA molecule may be distinguished by the hybridization withthe labeled probe.

It is possible to produce Ti-4 protein according to this invention in alarge scale, utilizing baculovirus expression system in which cDNAencoding the Ti-4 protein is inserted. An exemplary production methodwill be mentioned below. The Ti-4 protein is allowed to express in BmN4culture cells (silkworm cell) or in silkworm larvae, utilizing Bombyxmori nuclear polyhedral virus (BmNPV). The extract derived from theculture medium or from the body fluid of the silkworm larvae issubjected to chromatography to isolate the target protein.Alternatively, cDNA encoding the Ti-4 protein can be inserted intoAutographa californica nuclear polyhedral virus (AcNPV), and the Ti-4protein is allowed to express in Sf9 cells derived from Spodopterafrugiperda or in Tn5 cells derived from Trichoplusia ni. Supernatant ofthe culture medium is subjected to chromatography in the same manner toisolate the target protein.

The Ti-4 protein according to this invention can be produced in a largescale, utilizing an E. coli expression system in which cDNA encoding theTi-4 protein is inserted. For such a purpose, cDNA encoding the Ti-4protein can be amplified; and the cDNA can be inserted into a plasmidsuch as pMAL-c2g to construct a vector for expression of a fusionprotein with maltose binding protein (MBP). E. coli is transformed withthe expression vector, cultivated in a medium containing IPTG to induceexpression of the Ti-4 protein fused with MBP in the E. coli. The E.coli strains preferred for such a purpose include BL21 strain, forexample. The MBP fused Ti-4 protein induced in E. coli bodies can berecovered by disrupting the cell bodies. The MBP fused Ti-4 protein thusrecovered can be purified by affinity chromatography utilizing anamylose resin.

The Ti-4 protein of this invention having inhibitory effect on plateletaggregation can be produced in a large scale by; identification,isolation and purification of the active substance exhibiting inhibitoryeffect on platelet aggregation derived from salivary gland of ablood-sucking insect, cloning of the cDNA encoding the active substance,and expressing the protein in a baculovirus expression system. If theactive site responsible for the inhibitory effect on plateletaggregation will be determined on the protein and progression onstructural analysis of the protein will be achieved, the activesubstance will be produced through conventional synthetic procedureusing technique of molecular designing.

Moreover, the Ti-4 protein according to this invention will be quitevaluable as a prospective lead compound in the development of novelmedicines having an inhibitory effect on platelet aggregation.Furthermore, the structure of the Ti-4 protein can be modified invarious manners to develop novel compounds exhibiting higher inhibitorypotency on platelet aggregation. Thus, the Ti-4 protein according tothis invention provides a physiologically active substance that becomesthe basis for such investigation. Furthermore, it is quite useful as alead compound useful for further development of novel plateletaggregation inhibitors.

EXAMPLES

The present invention will be illustrated below by means of examples.However, the present invention is not limited in any way to thoseexamples.

Procedure for Gene Isolation

Salivary glands were removed from thorax of Triastoma infestans. ThemRNA was extracted from the salivary glands and purified using MicroprepmRNA purification kit (Amersham Pharmacia). Using the mRNA as atemplate, cDNA library of the salivary glands was constructed usingSuperscript Plasmid System (Life Technology). The clones (approximately550 clones in total) were randomly picked up from this library andplasmids were extracted and purified using QiAPrep Spin Miniprep kit(Qiagen). The base sequence of the cDNA inserted in the plasmid wasdetermined with ABI PRISM 310 genetic analyzer (PE Biosystems). Then theinventors analyzed on the base sequence of the cDNA thus determined,using a Genetyx ver. 8.5 (Software Development). As a result, it wasrevealed that four cDNA clones were possessing an identical basesequence, and it was designated to be Ti-4 gene.

Massive Expression of the Recombinant Protein and Purification of theProtein

The full length cDNA of Ti-4 gene was amplified by PCR and it wasinserted into BamHI restriction site of plasmid pAcYM1 to produce atransfer vector, and then it was purified using Plasmid Mini kit(Qiagen). The plasmid vector was introduced into insect culture cells(Sf-9) using Baculogold Linearized Baculovirus DNA (Phaemingen) toproduce recombinant baculoviruses. The recombinant viruses were allowedto infect another insect culture cells (Tn-5) and massive expression ofthe recombinant Ti-4 was achieved. The recombinant Ti-4 protein wassecreted into the culture medium and the recombinant protein waspurified via two steps of purification, that is, cation ion exchangechromatography with MONO S (Amersham Pharmacia) and gel-filtrationchromatography with TSK2000SW (Toso). The purified product was used inthe subsequent experiment.

Aggregation of Blood Platelets

Platelet aggregation was determined using washed human platelets byturbidimetric assay method. In other word, 50 μl of washed humanplatelets (6.0×10⁵/ml) was added into 40 μl of solution comprising thepurified Ti-4 protein dissolved into a buffer solution containing 50 mMTris-HCl (pH 7.4) and 150 mM NaCl, and the mixture was incubated at 37°C. After 10 minutes of incubation, 10 μl of collagen (Chronolog)solution (20 μg/ml) was added to it, and the mixture was furtherincubated for ten minutes. The light transmission of the solution wasmeasured at 600 nm using Micro Plate reader MPR-A4i (Toso).

FIG. 2 shows the effect of Ti-4 protein on collagen-induced plateletaggregation. FIG. 2 shows that the Ti-4 protein inhibits plateletaggregation in a dose dependent manner. This demonstrates that the Ti-4protein isolated from salivary gland of Triatoma infestans can inhibitcollagen-induced platelet aggregation.

The present invention provides Ti-4 protein, a novel protein derivedfrom Triatoma infestans, and a gene coding for the Ti-4 protein. TheTi-4 protein has an inhibitory activity on platelet aggregation,therefore, a medicine comprising the Ti-4 protein as an activeingredient will serve as a platelet aggregation inhibitor possiblyeffective for prevention and treatment of myocardial infarction,pulmonary infarction and cerebral infarction. Moreover, the Ti-4 proteinwill also serve as a highly prospective lead compound in the developmentof new platelet aggregation inhibitors.

1. An isolated and purified protein derived from Triatoma infestansconsisting of the amino acid sequence of following (a), or (b): (a) anamino acid sequence represented by amino acids Nos. −22-156 of SEQ IDNO:1; (b) an amino acid sequence exhibiting at least 95% homology withamino acid sequence (a), wherein amino acid sequence (b) inhibitsplatelet aggregation.
 2. An isolated and purified protein derived fromTriatoma infestans consisting of the amino acid sequence of following(a), or (b): (a) an amino acid sequence represented by amino acids Nos.1-156 of SEQ ID NO:1; (b) an amino acid sequence exhibiting at least 95%homology with amino acid sequence (a), wherein amino acid sequence (b)inhibits platelet aggregation.
 3. A platelet aggregation inhibitorcontaining the protein according to claim 2 as an active ingredient.